Characterization and DNA homology of Lactobacillus strains isolated from pig intestine

Twenty strains of the genus Lactobacillus were isolated from pig intestines and characterized with phenotypic tests. Eleven of these strains, together with selected reference strains, were subjected to DNA homology studies. Three major groups could be distinguished by biochemical/physiological tests and by DNA homology studies: one homofermentative group of the subgenus Thermobacterium and two heterofermentative groups. The DNA homology studies revealed that strains from the homofermentative group were related to Lactobacillus acidophilus strain VPI 1754, but showed a low relationship to the type strain of Lact. acidophilus . One group of the heterofermentative strains was related to the type strain of Lact. reuteri ; the other group, consisting of three strains, showed a low relationship to all reference strains used. These three strains had the unusual property of producing succinic acid in large amounts.     

Metabolism and some characteristics of lactobacilli isolated from the rumen of young calves

Fourteen strains of lactobacilli isolated from the rumen of young calves were studied to determine their biochemical characteristics, growth parameters, metabolism on lactose and sensitivity to 28 antimicrobial agents. Thirteen homofermentative strains belonged to Lactobacillus acidophilus and one heterofermentative strain resembled Lact. fermentum. The relevance of rumen lactobacilli to the nutrition of calves is discussed.     

Metabolism and some characteristics of lactobacilli isolated from the rumen of young calves

Fourteen strains of lactobacilli isolated from the rumen of young calves were studied to determine their biochemical characteristics, growth parameters, metabolism on lactose and sensitivity to 28 antimicrobial agents. Thirteen homofermentative strains belonged to Lactobacillus acidophilus and one heterofermentative strain resembled Lact. fermentum. The relevance of rumen lactobacilli to the nutrition of calves is discussed.     

Taxonomy of obligately homofermentative and facultatively heterofermentative lactobacilli in pig faeces

AIMS: Identification and characterization of obligately homofermentative and facultatively heterofermentative strains of Lactobacillus spp. isolated from the faeces of pigs that had been raised under different conditions. METHODS AND RESULTS: The phenotypic relatedness of the isolated strains and reference strains were determined by numerical analysis of total soluble cell protein patterns and simple physiological and biochemical tests. Of the 23 strains isolated from faeces, nine were obligately homofermentative and 14 facultatively heterofermentative. The strains clustered at r > or = 0.61 with Lactobacillus amylovorus (seven strains), Lactobacillus crispatus (one strain), Lactobacillus plantarum (14 strains) and Lactobacillus intestinalis (one strain). CONCLUSIONS: Results obtained from the physiological and biochemical tests confirmed the identity of the isolates as determined by numerical analysis of total soluble cell protein profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the association of Lact. crispatus and Lact. intestinalis with the gastro-intestinal tract of pigs.     

A survey of lactic acid bacteria in Italian silage

G razia , L. & S uzzi , G. 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56 , 373–379.
Lactic acid bacteria, isolated from Italian ensiled products, were represented by strains of the genera Lactobacillus and Leuconostoc . The predominant strains were heterofermentative lactobacilli, with Lactobacillus buchneri being the most frequent. Among homofermentative lactic acid bacteria, strains of Lact. plantarum and Lact. casei were recovered. Almost all strains utilized malic acid and showed good acid-tolerance, but only some of them were able to metabolize malic acid at extremely low pH; these were five homofermentative lactobacilli (4 Lact. plantarum and 1 Lacr. casei var. casei ) and two heterofermentative lactobacilli ( Lact. cellobiosus and Lactobacillus sp.).     

Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

AIMS: The overall growth kinetics of four potentially probiotic strains (Lactobacillus fermentum, Lact. reuteri, Lact. acidophilus and Lact. plantarum) cultured in malt, barley and wheat media were investigated. The objectives were to identify the main factors influencing the growth and metabolic activity of each strain in association with the cereal substrate. METHODS AND RESULTS: All fermentations were performed without pH control. A logistic-type equation, which included a growth inhibition term, was used to describe the experimental data. In the malt medium, all strains attained high maximum cell populations (8.10-10.11 log10 cfu ml(-1), depending on the strain), probably due to the availability of maltose, sucrose, glucose, fructose (approx. 15 g l(-1) total fermentable sugars) and free amino nitrogen (approx. 80 mg l(-1)). The consumption of sugars during the exponential phase (10-12 h) resulted in the accumulation of lactic acid (1.06-1.99 g l(-1)) and acetic acid (0.29-0.59 g l(-1)), which progressively decreased the pH of the medium. Each strain demonstrated a specific preference for one or more sugars. Since small amounts of sugars were consumed by the end of the exponential phase (17-43%), the decisive growth-limiting factor was probably the pH, which at that time ranged between 3.40 and 3.77 for all of the strains. Analysis of the metabolic products confirmed the heterofermentative or homofermentative nature of the strains used, except in the case of Lact. acidophilus which demonstrated a shift towards the heterofermentative pathway. All strains produced acetic acid during the exponential phase, which could be attributed to the presence of oxygen. Lactobacillus plantarum, Lact. reuteri and Lact. fermentum continued to consume the remaining sugars and accumulate metabolic products in the medium, probably due to energy requirements for cell viability, while Lact. acidophilus entered directly into the decline phase. In the barley and wheat media all strains, especially Lact. acidophilus and Lact. reuteri, attained lower maximum cell populations (7.20-9.43 log10 cfu ml(-1)) than in the malt medium. This could be attributed to the low sugar content (3-4 g l(-1) total fermentable sugar for each medium) and the low free amino nitrogen concentration (15.3-26.6 mg l(-1)). In all fermentations, the microbial growth ceased at pH values (3.73-4.88, depending on the strain) lower than those observed for malt fermentations, which suggests that substrate deficiency in sugars and free amino nitrogen contributed to growth limitation. CONCLUSIONS: The malt medium supported the growth of all strains more than barley and wheat media due to its chemical composition, while Lact. plantarum and Lact. fermentum appeared to be less fastidious and more resistant to acidic conditions than Lact. acidophilus and Lact. reuteri. SIGNIFICANCE AND IMPACT OF THE STUDY: Cereals are suitable substrates for the growth of potentially probiotic lactic acid bacteria.     

Lactobacilli isolated from sugary kefir grains capable of polysaccharide production and minicell formation

Homo- and heterofermentative species of Lactobacillus have been isolated from sugary kefir grains. Most of the homofermentative strains fermented tagatose and aldonitol and presented 48–54% of homology with Lactobacillus paracasei ssp. paracasei NCDO 151 (ex Lactobacillus casei ). The two variants of a heterofermentative species, although fermenting arabinose, were related to Lactobacillus hilgardii NCDO 264 (type strain) with 88% of homology. One of them produced polysaccharide from sucrose at pH 4–8 and 30°C; the best glucose conversion into polysaccharide was obtained from 3% of sucrose (81–8%), and the maximum production occurred about 35 hours after the end of the log phase of growth, in MRS sucrose broth. Polysaccharide formation did not occur above 40°C, a temperature at which no growth was observed. The two variants were forming minicells by abnormal divisions.     

Characterization and species recognition of Lactobacillus plantarum strains by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene

Twenty-one strains, labelled Lactobacillus plantarum or Lact. plantarum -like, and isolated from different natural sources, were characterized by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene using Hin dIII and Eco RI cleaved chromosomal DNA, together with Lact. plantarum ATCC 14917^T, Lact. pentosus ATCC 8041^T, Lact. plantarum ATCC 10776 and Lact. plantarum ATCC 8014. The fermentation patterns on API 50CH were recorded at 30°C and 37°C for all strains. The phenotypes were heterogeneous, and the ability to ferment 17 of the 49 carbohydrates varied. The fermentation of some carbohydrates, for example D-raffinose and D-arabitol, was temperature-dependent. Strains having identical API profiles were separated by the plasmid profile. All strains but one (affiliated to Lact. casei ) had identical 16S ribosomal DNA sequences ( Lact. plantarum/Lact. pentosus ). The RFLP study resulted in identical ribopatterns for 17 of the strains, including the type strain of Lact. plantarum (pattern A1). Four strains had related fragment patterns to that of Lact. plantarum sensu stricto; three of these strains had more than 60% DNA: DNA homology to the type strain of Lact. plantarum , and one had less than 50% DNA: DNA homology to Lact. plantarum ATCC 14917^T. Two strains had fragment patterns similar to the type strain of Lact. pentosus , and they had more than 80% DNA: DNA homology to Lact. pentosus ATCC 8041^T. One of the Lact. pentosus strains shared one band with the A1 pattern. The ribopatterns of Lact. plantarum were homogeneous (identical for 85% of the strains), irrespective of phenotype and source of isolation. RFLP of the 16S rRNA genes using Eco RI and Hin dIII might be used for species recognition of Lact. plantarum , but seems less suitable for strain typing.     

Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex

The Lactobacillus acidophilus complex includes Lact. acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. The objective of this work was to develop a rapid and definitive DNA sequence-based identification system for unknown isolates of the Lact. acidophilus complex. A approximately = 500 bp region of the 16S rRNA gene, which contained the V1 and V2 variable regions, was amplified from the isolates by the polymerase chain reaction. The sequence of this region of the 16S rRNA gene from the type strains of the Lact. acidophilus complex was sufficiently variable to allow for clear differentiation amongst each of the strains. As an initial step in the characterization of potentially probiotic strains, this technique was successfully used to identify a variety of unknown human intestinal isolates. The approach described here represents a rapid and definitive method for the identification of Lact. acidophilus complex members.     

Lactobacilli isolated from sugary kefir grains capable of polysaccharide production and minicell formation

Homo- and heterofermentative species of Lactobacillus have been isolated from sugary kefir grains. Most of the homofermentative strains fermented tagatose and aldonitol and presented 48-54% of homology with Lactobacillus paracasei ssp. paracasei NCDO 151 (ex Lactobacillus casei). The two variants of a hetero-fermentative species, although fermenting arabinose, were related to Lactobacillus hilgardii NCDO 264 (type strain) with 88% of homology. One of them produced polysaccharide from sucrose at pH 4.8 and 30 degrees C; the best glucose conversion into polysaccharide was obtained from 3% of sucrose (81.8%), and the maximum production occurred about 35 hours after the end of the log phase of growth, in MRS sucrose broth. Polysaccharide formation did not occur above 40 degrees C, a temperature at which no growth was observed. The two variants were forming minicells by abnormal divisions.     

Surface characteristics of lactobacilli isolated from human vagina

In the present paper, the taxonomic classification of 134 lactobacilli isolates from vaginal samples of 200 women of Tucumán, Argentina, is reported. They were clustered in three metabolic groups of the genus Lactobacillus, most belonging to the obligately homofermentative group (56%), mainly represented by Lactobacillus delbrueckii subsp. delbrueckii and L. acidophilus. In the facultatively heterofermentative group (24%), the dominant species were L. paracasei subsp. paracasei and L. agilis, and in the obligately heterofermentative group (20%), L. brevis was the dominant species. All strains were studied for surface characteristics and adhesion-predicting properties. A correlation between the methods employed for hydrophobicity testing of the different isolates (Microbial Adhesion to Hydrocarbons and Salt Aggregation Test) is reported. Most strains were highly hydrophobic. Their hemagglutination capability with human erythrocytes was also tested, which was positive only for a few strains. Some isolates were self-aggregating. From our results, strains that shared the properties assayed were selected for further testing of some other desirable characteristics, such as antagonistic substance production, adhesion to biological substrates, and appropriate technological properties, to suggest the elaboration of a probiotic for the vaginal tract.     

Preliminary characterization of microflora of Comté cheese

The evolution of the microflora of three Comté cheeses made in duplicate with raw milk from three different sources was followed during ripening. The same starter was used with each type of milk. The comparison of the cheeses did not reveal any significant difference in the development of the microflora. Starter lactic acid bacteria ( Streptococcus thermophilus and Lactobacillus helveticus) , which are added at the beginning of manufacture, decreased quickly in the first stages of ripening supporting the hypothesis of cell autolysis. Other micro-organisms, i.e. homofermentative and heterofermentative lactobacilli ( Lact. delbrueckii ssp. lactis , Lact. paracasei ssp. paracasei , Lact. rhamnosus and Lact. fermentum ), pediococci, enterococci and propionibacteria grew in cheese from small numbers in fresh curd. The characterization of Strep. thermophilus by pulsed-field gel electrophoresis showed that wild strains were also able to grow in the curd. The values for the genome size of 11 Strep. thermophilus strains determined in this investigation were in the range of 1·8–2·3 Mbp. The potential role of starter and raw milk microflora in cheese flavour development was considered.     

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

D. FIRA, M. KOJIC, A. BANINA, I. SPASOJEVIC, I. STRAHINIC AND L. TOPISIROVIC. 2001 . The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact. delbrueckii BGPF1, and Lact. acidophilus CH2 (Chr. Hansen's strain) and Lact. acidophilus V74 (Visby's strain), were compared. Results revealed that optimal pH for all four proteinases is 6·5, whereas temperature optimum varied among proteinases. Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both α_S1-casein and β-casein, showing very low activity towards κ-casein. The BGPF1 proteinase completely hydrolysed only β-casein. The BGRA43 proteinase completely hydrolysed all three casein fractions. The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases. DNA–DNA hybridization and PCR analysis showed that BGPF1 contains the prtB -like proteinase gene. Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.     

Specific enumeration of lactic acid bacteria in fermenting grape must and wine by colony hybridization with non-isotopic DNA probes

A. LONVAUD-FUNEL, A. JOYEUX AND O. LEDOUX. 1991. Total DNA extracted from lactic acid bacteria commonly found in musts and wines was randomly labelled with digoxigenin. It was assayed for the detection of several species by dot-blot hybridization. The method proved to be specific as there was no cross-hybridization between most of the species belonging to the genera Leuconostoc, Pediococcus and Lactobacillus , homofermentative and heterofermentative ( Lact. plantarum, Lact. casei, Leuc. mesenteroides, Leuc. oenos, Ped. damnosus, Ped. pentosaceus ). However, it failed for some Lact. brevis strains which strongly hybridized with Lact. hilgardii.
Colony hybridization was performed directly on plates soon after enumeration. Eight probes of the most common species were used; it was possible to follow the evolution of each species during the vinification of two red wines. According to the phase of alcoholic fermentation, then malolactic fermentation, the predominance or regression of bacilli and cocci could be established.     

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact. delbrueckii BGPF1, and Lact. acidophilus CH2 (Chr. Hansen's strain) and Lact. acidophilus V74 (Visby's strain), were compared. Results revealed that optimal pH for all four proteinases is 6.5, whereas temperature optimum varied among proteinases. Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both alphaS1-casein and beta-casein, showing very low activity towards kappa-casein. The BGPF1 proteinase completely hydrolysed only beta-casein. The BGRA43 proteinase completely hydrolysed all three casein fractions. The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases. DNA-DNA hybridization and PCR analysis showed that BGPF1 contains the prtB-like proteinase gene. Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.     

Lactose metabolism and lactase gene sequence homologies amongst lactobacilli

A number of strains of Lactobacillus spp., including the thermophilic and mesophilic dairy species, were screened for the presence of β -galactosidase ( β -gal) and phospho- β -galactosidase (pbg) enzyme activities. The majority of lactose fermenting strains exhibited β -gal rather than pbg enzyme activity with the highest levels in the thermophilic dairy species.
Correlation between these enzymes and the presence of specific genetic determinants was sought using probes for β -gal and pbg genes from Lactobacillus casei ssp. casei strain 64H. Southern transfer and filter hybridization showed that the β-gal probe shared homology with one strain of Lact. casei ssp. casei only. Sequences homologous to the pbg gene were detected only in plasmid DNA from the same strain of Lact. casei ssp. casei and with plasmid DNA from an apparently unrelated strain of Lactobacillus which exhibited no pbg activity. Two other strains of Lact. casei ssp. casei appeared to show homology between their chromosomal DNA and the pbg gene probe. No other homologies were detected. Therefore, although lactase activity could be detected in many strains of Lactobacillus spp., the genetic determinants involved did not share extensive homology.     

Lactic acid bacteria isolated from soy sauce mash in Thailand

Fourteen sphere-shaped and 30 rod-shaped lactic acid bacteria were isolated from soy sauce mash of two factories in Thailand. These strains were separated into two groups, Group A and Group B, by cell shape and DNA-DNA similarity. Group A contained 14 tetrad-forming strains, and these strains were identified as Tetragenococcus halophilus by DNA similarity. Group B contained 30 rod-shaped bacteria, and they were further divided into four Subgroups, B1, B2, B3, and B4, and three ungrouped strains by phenotypic characteristics and DNA similarity. Subgroup B1 contained 16 strains, and these strains were identified as Lactobacillus acidipiscis by DNA similarity. Subgroup B2 included two strains, and the strains were identified as Lactobacillus farciminis by DNA similarity. Subgroup B3 contained five strains. The strains had meso-diaminopimelic acid in the cell wall, and were identified as Lactobacillus pentosus by DNA similarity. The strains tested produced DL-lactic acid from D-glucose. Subgroup B4 contained four strains. The strains had meso-diaminopimelic acid in the cell wall, and they were identified as Lactobacillus plantarum by DNA similarity. Two ungrouped strains were homofermentative, and one was heterofermentative. They showed a low degree of DNA similarity with the type strains tested, and were left unnamed. The distribution of lactic acid bacteria in soy sauce mash in Thailand is discussed.     

A Genomic View of Lactobacilli and Pediococci Demonstrates that Phylogeny Matches Ecology and Physiology

Lactobacilli are used widely in food, feed, and health applications. The taxonomy of the genus Lactobacillus, however, is confounded by the apparent lack of physiological markers for phylogenetic groups of lactobacilli and the unclear relationships between the diverse phylogenetic groups. This study used the core and pan-genomes of 174 type strains of Lactobacillus and Pediococcus to establish phylogenetic relationships and to identify metabolic properties differentiating phylogenetic groups. The core genome phylogenetic tree separated homofermentative lactobacilli and pediococci from heterofermentative lactobacilli. Aldolase and phosphofructokinase were generally present in homofermentative but not in heterofermentative lactobacilli; a two-domain alcohol dehydrogenase and mannitol dehydrogenase were present in most heterofermentative lactobacilli but absent in most homofermentative organisms. Other genes were predominantly present in homofermentative lactobacilli (pyruvate formate lyase) or heterofermentative lactobacilli (lactaldehyde dehydrogenase and glycerol dehydratase). Cluster analysis of the phylogenomic tree and the average nucleotide identity grouped the genus Lactobacillus sensu lato into 24 phylogenetic groups, including pediococci, with stable intra- and intergroup relationships. Individual groups may be differentiated by characteristic metabolic properties. The link between phylogeny and physiology that is proposed in this study facilitates future studies on the ecology, physiology, and industrial applications of lactobacilli.     

Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction

Lactobacillus casei, Lact. paracasei and Lact. rhamnosus form a closely related taxonomic group within the heterofermentative lactobacilli. These three species are difficult to differentiate using traditional fermentation profiles. We have developed polymerase chain reaction primers which are specific for each of these species based on differences in the V1 region of the 16S rRNA gene. Sixty-three Lactobacillus isolates from cheese were identified using these primers. The 12 Lact. rhamnosus and 51 Lact. paracasei identified in this way were also differentiated using a randomly amplified polymorphic DNA (RAPD) primer.     

Characterization of natural isolate Lactobacillus acidophilus BGRA43 useful for acidophilus milk production

Lactobacillus acidophilus BGRA43 was selected from a set of human origin isolates of Lact. acidophilus strains for the highest growth rates and antagonistic effect against both Gram-positive and Gram-negative bacteria. The strain BGRA43 also exhibited an inhibitory effect on the growth of Clostridium sporogenes. Inhibition of this strain seems to be due to lactic acid production rather than hydrogen peroxide or bacteriocin. Growth of Lact. acidophilus BGRA43 in non-fat skim milk for 6 h at 37 °C resulted in a lowering of the pH value to 4·53. Besides the fast acidification, this strain generated a high viscosity of skim milk. These characteristics make the strain BGRA43 attractive for acidophilus milk production. Lactobacillus acidophilu s BGRA43 produces an extracellular proteinase. Whole cells efficiently degraded casein for 3 h at 37 °C especially α- and β-casein fractions. Total DNA isolated from the strain BGRA43 did not show any hybridization with lactococcal proteinase probes indicating that this strain produces a distinctive proteinase.